The use of kinase inhibitors to dissect and validate targetable nodes within cancer signaling pathways has revolutionized oncology drug discovery. Among the most interesting targets identified is CAMKK2, a kinase involved in a range of critical biological functions including metabolic homeostasis, protein synthesis, cell motility, gene transcription, cell survival, and macrophage activation. Substantial evidence demonstrates that CAMKK2 and the processes it regulates are involved in pathways of significant pathological importance in breast, prostate, hepatic, and gastric cancers. There is an immediate need for potent, selective, and in vivo active CAMKK2 chemical probes that can be used to define the roles of CAMKK2 in cell signaling and evaluate the therapeutic potential of CAMKK2 inhibitors in relevant models of human disease. Here we propose an iterative medicinal chemistry approach to develop CAMKK2 inhibitors that are potent and selective in vivo. We will use these new compounds to validate CAMKK2 as a viable therapeutic target for liver, breast, and prostate cancer. Using a unique compound design strategy, we have identified several potent and selective chemical leads that inhibit CAMKK2. We will optimize these leads into in vivo active CAMKK2 chemical probes using iterative medicinal chemistry (Aim 1). Aim 1 assays will focus on CAMKK2 potency against the isolated target, CAMKK2 potency in a cellular context, kinase selectivity, and optimization of properties to create molecules suitable for in vivo use. Our new selective CAMKK2 inhibitors will be evaluated in a range of disease-relevant cancer cellular models (Aim 2). These studies will explore the effects of CAMKK2 inhibition on cell proliferation, colony formation, macrophage activation, and on the remodeling of the tumor microenvironment. We will evaluate the efficacy of our optimized in vivo active CAMKK2 probes in mouse models of breast, prostate, and liver cancer (Aim 3). To accomplish our goals we have assembled a collaborative, multidisciplinary team with experience in kinase inhibitor optimization, CAMKK2 signaling, and tumor biology. Successful completion of this project will provide highly optimized CAMKK2-targeting molecules and may lead to new drugs for cancer treatment.